Project Summary Dehydroamino acids (?AAs) can increase the proteolytic stability of peptides as a result of a rigidifying effect caused by A1,3 strain that favors folded structures over random coil conformations. These residues should have great value to medicinal chemists and chemical biologists, but many types of ?AAs remain unexplored due to significant synthetic challenges. Accordingly, the objective of this proposal is to devise efficient routes to a range of ?AAs and then investigate the structures, stabilities, and potencies of peptides and natural products that contain them. The hypothesis is that new and efficient synthetic strategies will unlock access to a collection of ?AAs that can be used to tune the conformations, physical properties, and bioactivities of peptides. The rationale for this idea is that expanding the number of available ?AAs and defining their effects on peptide structure and function will provide new tools that will enable solutions to significant problems with relevance to human health. The hypothesis will be tested by pursuing three Specific Aims. Aim 1 involves expanding the realm of available ?,?-?AAs and devising new methods of incorporating them into peptides. Cyclic ?AAs and fluorinated ?AAs will be targeted. The new methodologies will include dehydrations and related eliminations that can be conducted on a solid support and are compatible with solid-phase peptide synthesis (SPPS). Aim 2 entails determining the impact of various types of ?,?-?AAs on peptide structure and stability as well as probing medicinal applications of peptides containing these residues. Secondary structures to be studied include turns, sheets, and helices. The inclusion of ?,?-?AAs in anticancer peptides and ?-sheet breaker peptides will be explored. Aim 3 consists of devising a robust synthesis of ?AAs containing the ?-thioenamide moiety and using it as part of an effort to determine the stereochemistry of the thioviridamide macrocycle. The thioviridamides are potent and selective anticancer peptides that contain the ?AA aminovinylcysteine. This residue will be constructed using an oxidative decarboxylative elimination that will be employed to construct 16 candidate structures of the thioviridamide macrocycle using SPPS. The approach is innovative because it upends the conventional wisdom stating that ?AAs are poorly suited to incorporation into bioactive peptides due to the perception that they are reactive to biological nucleophiles such as thiols. The significance of the proposed research lies in its ability to facilitate the use of peptides that contain ?AAs to solve important medicinal chemistry and chemical biology problems. Such studies could include the design of proteolytically stable peptides that are capable of disrupting protein?protein interactions with relevance to human diseases or the development of potent and stable analogs of the thioviridamides. This project is envisioned to raise the profile of ?AAs, which have been previously underutilized.